The presence of HIV-1 is determined by the presence of P24 viral antigen in an antigen capture assay or by detection of reverse transcriptase (RT) activity. The P24 assay, however, is about 200 fold more sensitive than the RT assay. Various laboratories have reported variable results in attempts to correlate P24 and RT activities. We have qualitatively and quantitatively analyzed the reaction requirements for RT activity and have increased the reaction rate (sensitivity) 500 fold. We find the RT activity now is directly proportional to enzyme (virus) concentration, reaction time and 32P TTP concentration. With the improved assay, we find a constant relationship between RT and P24 values. A cloned transformed cell line was established which produced defective Moloney murine leukemia virus containing a mutation of a single codon in the RNase H domain of reverse transcriptase. In vitro RT activity is unaffected by this mutation, whereas RNase H activity is 100 fold suppressed. Defective virus were used to infect NIH 3T3 cells, and minus strand DNA synthesis was determined by PCR amplification. After conditions were established to quantitate PCR results, we found initiation of viral DNA transcription to be a rare event: with cells infected with wild type M-MuLV, minus strand strong stop DNA was found within 15 min. These results suggest the mutation affects some prerequisite condition for initiation of RT in vivo but not in vitro with in homopolymer template-primers.